Abstract: For rapid discovery of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) main protease (M^pro) inhibitors from a natural product library, a novel colorimetric screening assay was developed. According to the colorimetric principle, the synthetic peptide TSAVLQ-para-nitroanilide (pNA) was used as the M^pro hydrolysis substrate. Subsequently, the working concentration of pNA substrate, M^pro working concentration, hydrolysis time and DMSO tolerance were optimized for the development of a simple and robust colorimetric screening assay. Through these systematic optimizations, we selected 0.4 μmol·L^-1 M^pro and 100 μmol·L^-1 pNA substrate as the optimal working concentrations in this colorimetric screening assay, and a high Z' factor of 0.9 was achieved. Using this screening assay, natural product ginkgolic acid C13:0 (GA13:0) was identified as a novel competitive M^pro inhibitor in vitro. Taken together, we have successfully developed a simple and optimized colorimetric screening assay, which will be vital for the discovery of novel SARS-CoV-2 M^pro inhibitors.